Preimplantation genetic diagnosis for spinal muscular atrophy type I

Abstract

Preimplantation genetic testing provides a means for couples at risk for spinal muscular atrophy type I to reduce their chance of initiating an affected pregnancy.

Molecular Diagnostics in Preimplantation Genetic Diagnosis

“…The second step involves the diagnostic test itself, which can be performed in a region of the IVF laboratory, an adjacent laboratory equipped to perform molecular analyses or in a completely separate dedicated molecular genetics laboratory equipped to process single cell sam- Various (gender determination to exclude hemizygotes) Nested PCR, agarose gel (ϩ/Ϫ X/Y) X-linked disorders 6 Various (gender determination to exclude hemizygotes) Nested PCR, heteroduplexing Cystic fibrosis 22,38,44,75 Tay-Sachs disease 111 3 bp deletion (⌬F508) 4 bp insertion Nested PCR, allele-specific amplification RhD blood typing 3 Myotonic dystrophy 127 ϩ/Ϫ RhD gene determines Rh status Expansion of (CTG)n trinucleotide repeat Nested PCR, restriction enzyme Cystic fibrosis, 23 Beta thalassemia, 83 Marfan syndrome, 107 Epidermolysis Bullosa, 100 Lesch-Nyhan syndrome, 101 Sickle cell anemia, 102 Fanconi's anemia, 103 Ornithine transcarbamylase deficiency, 104 Spinal muscular atrophy [108][109][110] Various point mutations Deletion. Distinguish between gene and pseudogene Nested PCR, restriction enzyme (2 mutations in 1 fragment)…”

“…Conversely, enzymes which digest the mutant but not the normal allele can usually be found, but in such cases incomplete or failed digestion could lead to an embryo being incorrectly diagnosed as normal. 33,[107][108][109] For such suboptimal assays the inclusion of an internal digestion control 29 could help to prevent a misdiagnosis (Figure 3). Enzyme digestion has also been an essential component of the preimplantation diagnosis of spinal muscular atrophy (SMA), in which a causative deletion in the survival motor neuron gene (SMN1) prevents PCR amplification of the mutant allele, but product from a highly homologous copy gene (SMN2) is specifically cut by the restriction enzyme Dra I.…”

“…Enzyme digestion has also been an essential component of the preimplantation diagnosis of spinal muscular atrophy (SMA), in which a causative deletion in the survival motor neuron gene (SMN1) prevents PCR amplification of the mutant allele, but product from a highly homologous copy gene (SMN2) is specifically cut by the restriction enzyme Dra I. 108,109 In these assays, no natu- Paternal mutation is a G-to-A transition which removes an existing Bfa I cut site (CTAG) preventing complete digestion of the 247-bp product (P, I). c and d: Direct sequencing of a purified PCR product from a compound heterozygous cell (I) using big dye terminators on an ABI 310 genetic analyzer.…”

Dificuldades diagnósticas na atrofia muscular espinhal

“…A extração do DNA foi feita a partir da camada de leucócitos de uma alíquota de aproximadamente 5 ml de sangue periférico, seguindo o protocolo adaptado de Lahiri e Nurnberger 4 . Para a amplificação dos éxons 7 e 8 do gene SMN foi utilizada a técnica de "Nested" de reação em cadeia da polimerase (PCR) a qual consiste na reamplificação do produto primário, que aumenta a sensibilidade e protege contra a contaminação que p o d e prejudicar o diagnostico 5 . Para cada reação de PCR foi p r eparada uma solução de 40 µl contendo: 10 mM Tris-HCl (pH 8,3), 50 mM KCl, 1,5 mM MgCl 2 , 1 µg de DNA genô-mico, 200 µM de dNTPs; 1 µM de cada iniciador; 0,03 IU/µl de Taq DNA Polimerase.O programa de amplificação referente à primeira etapa da PCR consistiu em uma desnaturação inicial a 96ºC por 8 minutos, seguido por 4 ciclos de desnaturação a 96ºC por 3 minutos, 1 minuto de pareamento a 52ºC, 2 minutos de extensão a 72ºC, seguido por 28 ciclos com o tempo de desnaturação diminuído para 1 minuto e extensão final de 59 minutos a 72ºC.…”

Multiplex Nested PCR for Preimplantation Genetic Diagnosis of Spinal Muscular Atrophy

“…PGD requires in vitro fertilization (IVF) and single-cell analysis of one or two blastomeres obtained by embryo biopsy at the 6-to 8-cell stage, followed by transfer of unaffected embryos [10,11]. Several protocols for PGD of SMA have previously been reported [12][13][14][15][16]. These have relied on the detection of homozygous deletion of exon 7 or 8 of the SMN1 gene by either two rounds of nested PCR followed by restriction enzyme digestion [13,14,16] or by a single round of fluorescent PCR using the amplification refractory mutation system (ARMS) [15].…”